Dataset Information

Rapid differentiation of Mycobacterium tuberculosis and M. bovis by high-resolution melt curve analysis.

ABSTRACT: Identification and characterization of the Mycobacterium tuberculosis strains are important for clinical and therapeutic management of tuberculosis. Real-time PCR with a high-resolution melt assay was found to improve the diagnostic process. The assay includes differentiation between M. tuberculosis and Mycobacterium bovis based on one single-nucleotide polymorphism (SNP) in the narGHJI and oxyR genes and determination of M. bovis based on the region of differences 1 (RD1). This assay correctly identified the 7 tested Mycobacterium reference strains and 52 clinical samples with a sensitivity of 2 pg DNA. This assay will help in prescribing adequate treatment and monitoring disease dynamics.

SUBMITTER: Ereqat S

PROVIDER: S-EPMC3020821 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Rapid differentiation of Mycobacterium tuberculosis and M. bovis by high-resolution melt curve analysis.

Ereqat Suheir S Bar-Gal Gila Kahila GK Nasereddin Abedelmajeed A Azmi Kifaya K Qaddomi Sharif E SE Greenblatt Charles L CL Spigelman Mark M Abdeen Ziad Z

Journal of clinical microbiology 20100915 11

Identification and characterization of the Mycobacterium tuberculosis strains are important for clinical and therapeutic management of tuberculosis. Real-time PCR with a high-resolution melt assay was found to improve the diagnostic process. The assay includes differentiation between M. tuberculosis and Mycobacterium bovis based on one single-nucleotide polymorphism (SNP) in the narGHJI and oxyR genes and determination of M. bovis based on the region of differences 1 (RD1). This assay correctly ...[more]

PMID: 20844219

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Project description:The possibility of introducing a reliable assay for a quick identification and differentiation of the main species of Mycobacterium tuberculosis complex (MTBC) supports the improvement of efficient tuberculosis combating strategies worldwide. Commercially available assays are often based on cultured samples; however, due to the long cultivation time of mycobacteria, results are delayed. Developed PCR approaches have been published previously, though, when testing intricate veterinary samples, the complex composition of multiplex qPCRs frequently leads to assay failure. In order to overcome those limits, a paradigm of a three-reaction high-resolution melting (HRM) assay for the simultaneous identification and differentiation of the main members of MTBC was established. The assay is based on single nucleotide polymorphisms within gyrB and gyrA, which have been used as target for the establishment of two highly specific HRM assays (HRM assays 1 and 2) discriminating M. tuberculosis/ Mycobacterium canetti, Mycobacterium bovis/M. bovis BCG, Mycobacterium caprae/rare M. caprae/M. bovis ecotypes, Mycobacterium africanum/Mycobacterium orygis/ Mycobacterium pinnipedii/Clade A1, Mycobacterium microti, and a rare subtype of M. canettii followed by a third HRM assay (HRM assay 3) allowing a further differentiation of M. bovis, M. bovis BCG, and a rare subtype of M. caprae/M. bovis, which is considered to be a novel ecotype. High-resolution melting assay 1 is described in a previously published report. High-resolution melting assay 2 showed 100% correlation of all 39 examined isolates with the results of a commercial identification kit. 96% of the clinical samples tested demonstrated concordant results. High-resolution melting assay 3 showed an accordance of 100% with the results of the commercially available identification kit of all 22 samples analyzed. The proposed strategy of the three-reaction HRM assay can be used for an accurate differentiation of up to seven groups of MTBC and potentially to identify a rare subtype of M. canettii either on isolates or on clinical samples.

Project description:The emergence of Mycobacterium abscessus complex (MABC) infection is the most noteworthy health care problem. Clarithromycin (CLA) and amikacin (AMK) constitute the cornerstone of treatment for patients infected with MABC; thus, early detection of resistance to these two drugs is essential for formulating effective therapeutic regimens. In the present study, we aimed to validate the use of MeltPro MAB assay, a melting curve analysis with dually labeled probes, on a set of clinical isolates to detect CLA and AMK resistance. A total of 103 clinical MABC strains were collected in our analysis, including 76 strains of M. abscessus subsp. Abscessus (MAA) and 27 strains of M. abscessus subsp. Massiliense (MAM). In vitro susceptibility testing revealed that two isolates exhibited intrinsic CLA resistance by harboring A2270T mutation in rrl, and inducible resistance was noted in 42 isolates. Additionally, two MAA isolates with erm(41)T28 genotype were susceptible to CLA. Notably, we found three out of 44 isolates had two melting curve peaks, representing the simultaneous presence of mutant and the wild type in these specimens. In contrast, no known mutations were identified in six AMK-resistant isolates. Further analysis revealed that MeltPro yielded 100% and 96.67% sensitivity and specificity for detecting CLA resistance. In summary, this study firstly demonstrates that MeltPro is a promising diagnostic for early detection of CLA resistance for MABC isolates, which significantly improves the turnaround time within 2 h. Approximate two fifths of MABC isolates are resistant to CLA by 23S rRNA mutation or its methylation, emphasizing the urgent need for early detection of CLA resistance prior to empirical treatment of MABC infections. IMPORTANCE Mycobacterium abscessus complex (MABC) has attracted increasing attention due to the numerous cases of infection. This pathogen is notorious for its intrinsic drug resistance, which complicates clinical management of patients with MABC infections. Clarithromycin (CLA) and amikacin (AMK) are the cornerstone of treatment regimens for MABC. Herein, our data firstly demonstrates that MeltPro is a promising diagnostic for early detection of CLA resistance for MABC isolates. The high frequency of CLA-resistant MABC isolates in China emphasizes the urgent need for early detection of CLA resistance prior to empirical treatment of MABC infections.

Dataset Information

Rapid differentiation of Mycobacterium tuberculosis and M. bovis by high-resolution melt curve analysis.

Publications

Rapid differentiation of Mycobacterium tuberculosis and M. bovis by high-resolution melt curve analysis.

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